Hyperphosphorylation of tau is a hallmark of tauopathies, with specific phosphorylation sites elevated in pathological fibrils. Yet, the molecular role of this post-translational modification (PTM) in driving tau aggregation remains unclear. Tau proteins assemble in register, placing high-phosphoryl groups ~4.8 Å apart for high abundance PTMs, requiring an energetically favorable arrangement. This study tests the hypothesis that phosphoryl groups within the fibril core-forming segment readily associate into an extended “wire” that stabilizes the amyloid fibril, counter to the common assumption that closely packed phosphoryl groups is energetically unfavorable due to electrostatic repulsion. We examined two phosphorylation sites linked to neurodegeneration, serine 305 (S305^p) and tyrosine 310 (Y310^p), using seeding-competent fibrils of the tau peptide jR2R3-P301L. Multiple-quantum spin counting (MQ-SC) by ³¹P solid-state NMR with dynamic nuclear polarization revealed at least six phosphorus spins arranged in 1D within a protofibril, consistent with the observed MQ coherence order of four. Molecular dynamics simulations and 2D ¹H-³¹P heteronuclear correlation NMR and revealed water-mediated phosphoryl wires enhancing the stability and seeding competency of fibrils made of S305^p-phosphorylated jR2R3-P301L compared to the unmodified one. This work introduces the concept that phosphorylation within tau’s core can promote fibril registry and stability through water-mediated hydrogen-bonded phosphoryl wires.

-Han Lab